Forkhead Box O3a Transcription Factor Research Articles

Toxoplasma gondii inhibits the expression of autophagy-related genes through AKT-dependent inactivation of the transcription factor FOXO3a.

The intracellular parasite Toxoplasma gondii induces host AKT activation to prevent autophagy-mediated clearance; however, the molecular underpinnings are not fully understood. Autophagy can be negatively regulated through AKT-sensitive phosphorylation and nuclear export of the transcription factor Forkhead box O3a (FOXO3a). Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hinders host autophagy through AKT-dependent inactivation of FOXO3a. We found that infection by type I and II strains of T. gondii promotes gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in human foreskin fibroblasts (HFF) and murine 3T3 fibroblasts. Mechanistically, AKT-sensitive phosphorylation of FOXO3a by T. gondii required live infection and the activity of PI3K but was independent of the plasma membrane receptor EGFR and the kinase PKCα. Phosphorylation of FOXO3a at AKT-sensitive residues was paralleled by its nuclear exclusion in T. gondii-infected HFF. Importantly, the parasite was unable to drive cytoplasmic localization of FOXO3a upon pharmacological blockade of AKT or overexpression of an AKT-insensitive mutant form of FOXO3a. Transcription of a subset of bona fide autophagy-related targets of FOXO3a was reduced during T. gondii infection in an AKT-dependent fashion. However, parasite-directed repression of autophagy-related genes was AKT-resistant in cells deficient in FOXO3a. Consistent with this, T. gondii failed to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the parasitophorous vacuole upon chemically or genetically induced nuclear retention of FOXO3a. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing. IMPORTANCE The parasite Toxoplasma gondii is the etiological agent of toxoplasmosis, an opportunistic infection commonly transmitted by ingestion of contaminated food or water. To date, no effective vaccines in humans have been developed and no promising drugs are available to treat chronic infection or prevent congenital infection. T. gondii targets numerous host cell processes to establish a favorable replicative niche. Of note, T. gondii activates the host AKT signaling pathway to prevent autophagy-mediated killing. Herein, we report that T. gondii inhibits FOXO3a, a transcription factor that regulates the expression of autophagy-related genes, through AKT-dependent phosphorylation. The parasite's ability to block the recruitment of the autophagy machinery to the parasitophorous vacuole is impeded upon pharmacological inhibition of AKT or overexpression of an AKT-insensitive form of FOXO3a. Thus, our study provides greater granularity in the role of FOXO3a during infection and reinforces the potential of targeting autophagy as a therapeutic strategy against T. gondii.

Parkin-mediated mitophagy is negatively regulated by FOXO3A, which inhibits Plk3-mediated mitochondrial ROS generation in STZ diabetic stress-treated pancreatic β cells.

Diabetes mellitus (DM) is one of the most researched metabolic diseases worldwide. It leads to extensive complications such as cardiovascular disease, nephropathy, retinopathy, and peripheral and central nervous system through an inability to produce or respond to insulin. Although oxidative stress-mediated mitophagy has been reported to play an important role in the pathogenesis of DM, specific studies are still lacking as well as remain highly controversial. Here, we found that Parkin-mediated mitophagy in pancreatic β cells under streptozotocin (STZ)-diabetic stress was induced by Polo-like kinase 3 (Plk3) and inhibited by the transcription factor Forkhead Box O3A (FOXO3A). STZ stress induces mitochondrial recruitment of Parkin through Plk3-mediated mitochondrial reactive oxygen species (ROS) generation, which causes pancreatic cell damage. Conversely, FOXO3A acts as negative feedback to prevent diabetic stress by inhibiting Plk3. Meanwhile, antioxidants including N-acetylcysteine (NAC) and natural COA water scientifically block these mitochondrial ROS and mitochondrial recruitment of Parkin by inhibiting Plk3. Through a 3D organoid ex vivo model, we confirmed that not only ROS inhibitors but also mitophagy inhibitory factors such as 3-MA or Parkin deletion can compensate for pancreatic cell growth and insulin secretion under STZ diabetic stress. These findings suggest that the Plk3-mtROS-PINK1-Parkin axis is a novel mitophagy process that inhibits pancreatic β-cell growth and insulin secretion and FOXO3A and antioxidants may provide new alternatives for effective diabetes treatment strategies in the future.

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

Association of Forkhead Box O3a (FOXO3a) single – nucleotide polymorphism with bronchial asthma in Egyptian population

Asthma is a common chronic inflammatory condition with a highly complex genetic predisposition and environmental factors which play an important role in its development. Polymorphism in FOXO3a transcription factor has been linked to a number of inflammatory and respiratory diseases such as bronchiolitis and idiopathic pulmonary fibrosis suggesting that it may be implicated in the pathogenesis of asthma. This study aimed to investigate FOXO3a SNP (rs13217795) association with bronchial asthma and its degree of severity in adult Egyptian population. This case control study included 60 asthmatic patients and 40 apparently healthy controls. Peripheral blood samples were collected from all participants. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The study revealed high frequency of the mutant TT genotype of FOXO3a gene in asthma patients (51.7%) than controls (12.5%) with OR= 7.48 & 95% CI (2.58-21.71) (P˂0.05) and in severe cases (41.9%) compared to mild and moderate cases (25.8% and12.5%, respectively). T allele frequency showed significant statistical association with asthma, OR= 12.40, 95% CI (5.65-27.19) (P˂0.05). However, there was no association between T allele and disease severity. The high frequency of the mutant TT genotype among patients and sever cases may indicates that FOXO3a rs13217795 C>T single nucleotide polymorphism can be considered as a risk factor in development and severity of asthma.

MO629XANTHINE OXIDASE INHIBITOR ATTENUATES RENAL OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION THROUGH THE INHIBITION OF VEGF-NADPH OXIDASES IN DIABETIC NEPHROPATHY

Abstract Background and Aims Xanthine oxidase (XO) is one of major source of reactive oxygen species, and a XO inhibitor, febuxostat has been reported to the protection of kidney diseases. We investigated whether febuxostat exerts renoprotective effects against diabetic nephropathy (DN). Method Eight-week Male C57BL/6 mice were divided into four groups: Control group (Cont), Febuxostat control group (FEB), streptozotocin treated group (STZ) and a febuxostat and STZ-treated diabetes group (STZ+FEB). STZ was used to induce diabetes (50 mg/kg/day, 5 days), and 5 mg/kg of febuxostat was treated to experimental mice for 8 weeks. Results STZ-treated diabetic mice were significantly decreased in serum and kidney XO levels, serum cystatin C and albuminuria by febuxostat treatment. Febuxostat treatment decreased renal hypertrophy and mesangial matrix expansion in STZ-treated diabetic mice. Febuxostat treatment suppressed the expression of vascular endothelial growth factor (VEGF)1 and 3, NADPH oxidase (NOX)1, 2, and 4, and the levels of their catalytic subunit mRNA in in STZ-treated diabetic mice. Febuxostat treatment was accompanied by the downregulation of Akt phosphorylation, followed by the suppression of transcription factor forkhead box O3a phosphorylation and the enhancement of endothelial nitric oxide synthase. Finally, febuxostat improved oxidative stress and resulted in decreased 8-hydroxy-2'-deoxyguanosine and kidney malondialdehyde levels, and increased superoxide dismutase activity in STZ-treated diabetic mice. Conclusions Febuxostat attenuated DN by modulating oxidative stress and endothelial function through VEGF–NADPH oxidase signaling pathway.

Induction of foxo3a protects turtle neurons against oxidative stress

The detrimental effects of oxidative stress caused by the accumulation of Reactive Oxygen Species (ROS) factor into aging, senescence and several neurodegenerative diseases. Mammalian models are extremely susceptible to the stresses that follow the restoration of oxygen after anoxia; however some organisms including the freshwater turtle Trachemys scripta can withstand extended anoxia and reoxygenation without apparent pathology. The ability of the turtle to withstand these conditions is thought to be linked to the upregulation of protective mechanisms such as heat shock proteins (HSP) as well as the suppression of ROS formation and the upregulation of antioxidant defenses. One such antioxidant mechanism is the transcription factor Forkhead box O3a (FOXO3a), that has been shown to be activated in several animal models during oxidative stress. In this study, we utilized both the transfection of a plasmid carrying foxo3a and the pharmacological manipulation of foxo3a using the green tea extract Epigallocatechin-3-gallate (EGCG) to investigate the protective role of FOXO3a in the turtle brain. Our studies found that transcript levels of foxo3a were upregulated significantly during reoxygenation with greater increases during chemical oxidative stress. Induction of foxo3a by direct transfection significantly decreased cell death during chemical oxidative stress. Cells treated with EGCG also showed increased foxo3a expression and decreased cell death in the presence of H2O2. These results agree with results seen in other animal models and suggest that EGCG (through the upregulation of foxo3a) may be a therapeutic target against oxidative stress damage that warrants further investigation.

Forkhead Box O3a requires BAF57, a subunit of chromatin remodeler SWI/SNF complex for induction of p53 up-regulated modulator of apoptosis(Puma) in a model of Parkinson's disease.

Parkinson's disease (PD) results from the selective loss of dopaminergic neurons of substantia nigra pars compacta region of the midbrain. It has been reported that the transcription factor forkhead Box O3a (FoxO3a) is activated and induces pro-apoptotic protein such as Bcl-2-interacting mediator of cell death (BIM) and p53 up-regulated modulator of apoptosis (PUMA) in variety of neuron death paradigms. Activity of FoxO3a is governed by its post-translational modifications which control its subcellular localization. Aim of this study was to determine whether FoxO3a is activated and up-regulates its pro-apoptotic genes to induce neuron death in PD. We exposed neuronal PC12 cells or primary cultures of dopaminergic neurons to 6-hydroxy dopamine (6-OHDA) and infused 6-OHDA in rat brain to develop PD models. We found that FoxO3a undergoes multiple post-translational modifications which render its nuclear localization in dopaminergic neuronal cells in response to 6-OHDA. The nuclear redistribution of FoxO3a is significantly increased in dopaminergic neurons of 6-OHDA infused rat brains as well. Moreover, FoxO3a is required for dopaminergic neurodegeneration in response to 6-OHDA as RNAi-mediated silencing of FoxO3a protects these cells from 6-OHDA toxicity. In a search of the downstream targets we identified PUMA as a direct target of FoxO3a. By knocking down FoxO3a we could successfully block the up-regulation of the pro-apoptotic protein PUMA in this model. Recently, it has been reported that chromatin remodeler SWItch/sucrose non-fermentable binds to FOXO and activates transcription. We found that Brg-associated factor 57 (BAF57), a subclass of SWItch/sucrose non-fermentable is up-regulated and play a necessary role in neuron death induced by 6-OHDA. Moreover, it is required for induction of PUMA by FoxO3a in this cellular model of PD. Taken together, our study suggest that FoxO3a is activated, translocates to nucleus, induces its pro-apoptotic target PUMA in the presence of chromatin remodeler BAF57 to execute neuron death in cellular models of PD.

FoxO3a depletion accelerates cutaneous wound healing by regulating epithelial‑mesenchymal transition through β‑catenin activation.

The hysteresis of keratinocyte (KC) re-epithelialization is an important factor resulting in chronic wounds; however, the molecular mechanisms involved in this cellular response remain yet to be completely elucidated. The present study demonstrated the function of transcription factor Forkhead box O3a (FoxO3a) in KC growth and migration functional effects, resulting in restrained KC re-epithelialization during wound healing. In chronic wound tissue samples, the expression of FoxO3a was significantly increased when compared with the acute wound healing group (P<0.01). Overexpressing FoxO3a significantly inhibited, whereas silencing endogenous FoxO3a enhanced, the growth and migration of HaCaT cells in vitro. Further investigation revealed that FoxO3a negatively regulated matrix metalloproteinases 1 and 9, and increased the expression of tissue inhibitor of metalloproteinase 1. In addition, the upregulation of FoxO3a retarded, whereas the downregulation of FoxO3a accelerated, transforming growth factor-β1-induced epithelial-mesenchymal transition in HaCaT cells. Mechanistically, the overexpression of FoxO3a inactivated β-catenin signaling and markedly reduced the levels of nuclear β-catenin. These results reveal a novel mechanism of FoxO3a in regulating KC re-epithelialization, and provide novel targets for the prevention and treatment of chronic wounds.

Effects of tanshinones mediated by forkhead box O3a transcription factor on the proliferation and apoptosis of lung cancer cells.

According to global cancer statistics in 2012, lung cancer (LC) was the most frequently diagnosed cancer and the leading cause of cancer-associated mortality among males worldwide. Owing to the limited therapeutic approaches available, novel methods for treating LC are required. Tanshinones (Ts) have previously been proved to be effective in treating cardiovascular disease, inflammatory disease and cancer, and have been reported to regulate cell proliferation and apoptosis of LC. The underlying molecular mechanism of action of Ts remains unclear. Furthermore, forkhead box O3a (FoxO3a) has been reported to be a critical gene in cell apoptosis. Therefore, the A549 lung cancer cell line was transfected with FoxO3a small interfering RNA (siRNA) or scrambled siRNA, and the cells which exhibited the most successful transfection efficacy were selected for further investigation into the underlying molecular mechanism of the influence of Ts on FoxO3a in LC cells. Various concentrations of Ts were assigned to experimental groups I-IV (5, 10, 20 and 30 µmol/l Ts, respectively). An MTT assay revealed that Ts inhibited cell proliferation in a dose- and time-dependent manner compared with the control group (CON; without Ts administration) with a maximal dose of 20 µmol/l at 72 h treatment (P<0.05). Similarly, compared with CON, flow cytometry results revealed that Ts induce LC cell apoptosis in a dose-dependent manner (P<0.05). Consistently, the expression levels of FoxO3a mRNA and protein were restored following treatment with Ts in a dose-dependent manner, alongside caspase-3 activation. On the basis of these results, we hypothesize that Ts regulate LC cell proliferation and apoptosis by triggering an apoptotic cascade through the FoxO3a/caspase-3 signaling pathway.

HDAC4 regulates vascular inflammation via activation of autophagy.

Angiotensin II (Ang II) causes vascular inflammation, leading to vascular endothelial cell dysfunction, and is associated with the development of cardiovascular diseases. Therefore, interventions in inflammation may contribute to the reduction of cardiovascular diseases. Here, we aim to demonstrate that HDAC4, one of class IIa family histone de-acetylases (HDACs) members, promotes autophagy-dependent vascular inflammation. By loss-of-function approaches, our study provides the first evidence that HDAC4 mediates Ang II-induced vascular inflammation in vitro and in vivo. In response to the Ang II, HDAC4 expression is up-regulated rapidly, with increased autophagic flux and inflammatory mediators in vascular endothelial cells (VECs). In turn, HDAC4 deficiency suppresses activation of autophagy, leading to reduced inflammation in Ang II-induced VECs. Consistently, using autophagy inhibitor or silencing LC3-II also alleviates vascular inflammation. Furthermore, HDAC4 regulates autophagy via facilitating transcription factor forkhead box O3a (FoxO3a) de-acetylation, thereby to increase its transcriptional activity. Loss of HDAC4 in VECs results in inhibition of FoxO3a de-acetylation to block its transcriptional activity, leading to downregulation of the downstream FoxO3a target, and hence reduces autophagy and vascular inflammation. FoxO3a silencing using siRNA approach significantly inhibits activation of autophagy. Finally, knockdown of HDAC4 in Ang II-infused mouse models ameliorates vascular inflammation, suggesting that inhibitor of HDAC4 may be potential therapeutics for vascular diseases associated with inflammation. These results suggest that HDAC4-mediated FoxO3a acetylation regulates Ang II-induced autophagy activation, which in turn plays an essential role in causing vascular inflammation.

Levels of α‐ and β‐synuclein regulate cellular susceptibility to toxicity from α‐synuclein oligomers

α-Synuclein (α-syn) is associated with a range of diseases, including Parkinson disease. In disease, α-syn is known to aggregate and has the potential to be neurotoxic. The association between copper and α-syn results in the formation of stellate toxic oligomers that are highly toxic to cultured neurons. We further investigated the mechanism of toxicity of α-syn oligomers. Cells that overexpress α-syn showed increased susceptibility to the toxicity of the oligomers, while those that overexpressed β-syn showed increased resistance to the toxic oligomers. Elevated α-syn expression caused an increase in expression of the transcription factor Forkhead box O3a (FoxO3a). Inhibition of FoxO3a activity by the overexpression of DNA binding domain of FoxO3a resulted in significant protection from α-syn oligomer toxicity. Increased FoxO3a expression in cells was shown to be caused by increased ferrireductase activity and Fe(II) levels. These results suggest that α-syn increases FoxO3a expression as a result of its intrinsic ferrireductase activity. The results also suggest that FoxO3a plays a pivotal role in the toxicity of both Fe(II) and toxic α-syn species to neuronal cells.-Angelova, D. M., Jones, H. B. L., Brown, D. R. Levels of α- and β-synuclein regulate cellular susceptibility to toxicity from α-synuclein oligomers.

Nuclear forkhead box O3a accumulation contributing to the proliferative suppression in liver cancer cells by PI3K/Akt signaling pathway.

Serine/threonine kinase is originally identified as an oncogene and the forkhead box transcription factor forkhead box O3a (Foxo3a) has been found to be decreased in various human cancers. In the present study, we explored the expression of Akt and FOXO3a in liver cancer cells. Akt level was detected by Western blotting analysis. Cell viability of HepG2, MHCC-97H, Bel7402, and L02 was determined by MTT assay. FoxO3a level was determined by Western blotting analysis. Akt level was significantly higher in liver cancer cell lines HepG2 and MHCC97-H, compared with the immortalized liver cell line L02. MTT assay results demonstrated that LY294002 significantly suppressed cell proliferation of HepG2 and MHCC-97H cells in a dose- and time-dependent manner. The underlying molecular mechanism was that miR-370 inhibited cell proliferation of liver cancer cells by activating FoxO3a. PI3K inhibitor decreased the levels of phosphorylated FOXO3a and increased the levels of nuclear FOXO3a. It also inhibited cell proliferation of liver cancer cells partly by PI3K/Akt/FOXO3a signaling pathways.

Forkhead Box O3A (FOXO3) and the Mitochondrial Disulfide Relay Carrier (CHCHD4) Regulate p53 Protein Nuclear Activity in Response to Exercise

Although exercise is linked with improved health, the specific molecular mechanisms underlying its various benefits require further clarification. Here we report that exercise increases the nuclear localization and activity of p53 by acutely down-regulating coiled-coil-helix-coiled-coil-helix domain 4 (CHCHD4), a carrier protein that mediates p53 import into the mitochondria. This response to exercise is lost in transgenic mice with constitutive expression of CHCHD4. Mechanistically, exercise-induced nuclear transcription factor FOXO3 binds to the CHCHD4 promoter and represses its expression, preventing the translocation of p53 to the mitochondria and thereby increasing p53 nuclear localization. The synergistic increase in nuclear p53 and FOXO3 by exercise can facilitate their known interaction in transactivating Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase that mediates adaptation to various stresses. Thus, our results reveal one mechanism by which exercise could be involved in preventing cancer and potentially other diseases associated with aging.

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

Phosphoinositides: Two-Path Signaling in Neuronal Response to Oligomeric Amyloid β Peptide.

We have previously demonstrated that oligomeric amyloid β peptide (oAβ) together with iron overload generates synaptic injury and activation of several signaling cascades. In this work, we characterized hippocampal neuronal response to oAβ. HT22 neurons exposed to 500nM oAβ showed neither increased lipid peroxidation nor altered mitochondrial function. In addition, biophysical studies showed that oAβ did not perturb the lipid order of the membrane. Interestingly, although no neuronal damage could be demonstrated, oAβ was found to trigger bifurcated phosphoinositide-dependent signaling in the neuron, on one hand, the phosphorylation of insulin receptor, the phosphatidylinositol 3-kinase (PI3K)-dependent activation of Akt, its translocation to the nucleus and the concomitant phosphorylation, inactivation, and nuclear exclusion of the transcription factor Forkhead Box O3a (FoxO3a), and on the other, phosphoinositide-phospholipase C (PI-PLC)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Pharmacological manipulation of the signaling cascades was used in order to better characterize the role of oAβ-activated signals, and mitochondrial function was determined as a measure of neuronal viability. The inhibition of PI3K, PI-PLC, and general phosphoinositide metabolism impaired neuronal mitochondrial function. Furthermore, increased oAβ-induced cell death was observed in the presence of phosphoinositide metabolism inhibition. Our results allow us to conclude that oAβ triggers the activation of phosphoinositide-dependent signaling, which results in the subsequent activation of neuroprotective mechanisms that could be involved in the determination of neuronal fate.

The Neuroprotective Effect of Klotho is Mediated via Regulation of Members of the Redox System

Generation of reactive oxygen species (ROS), leading to oxidative damage and neuronal cell death, plays an important role in the pathogenesis of neurodegenerative disorders, including Alzheimer disease. The present study aimed to examine the mechanism by which the anti-aging protein Klotho exerts neuroprotective effects against neuronal damage associated with neurodegeneration and oxidative stress. Pretreatment of rat primary hippocampal neurons and mouse hippocampal neuronal cell line HT22 with recombinant Klotho protected these cells from glutamate and oligomeric amyloid β (oAβ)-induced cytotoxicity. In addition, primary hippocampal neurons obtained from Klotho-overexpressing mouse embryos were more resistant to both cytotoxic insults, glutamate and oAβ, compared with neurons from wild-type littermates. An antioxidative stress array analysis of neurons treated with Klotho revealed that Klotho significantly enhances the expression of the thioredoxin/peroxiredoxin (Trx/Prx) system with the greatest effect on the induction of Prx-2, an antioxidant enzyme, whose increase was confirmed at the mRNA and protein levels. Klotho-induced phosphorylation of the PI3K/Akt pathway, a pathway important in apoptosis and longevity, was associated with sustained inhibitory phosphorylation of the transcription factor forkhead box O3a (FoxO3a) and was essential for the induction of Prx-2. Down-regulation of Prx-2 expression using a lentivirus harboring shRNA almost completely abolished the ability of Klotho to rescue neurons from glutamate-induced death and significantly, but not completely, inhibited cell death mediated by oAβ, suggesting that Prx-2 is a key modulator of neuroprotection. Thus, our results demonstrate, for the first time, the neuroprotective role of Klotho and reveal a novel mechanism underlying this effect.

Tiny transporters: how exosomes and calcineurin signaling regulate miR-23a levels during muscle atrophy. Focus on “miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export”

Editorial FociTiny transporters: how exosomes and calcineurin signaling regulate miR-23a levels during muscle atrophy. Focus on “miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export”Christopher S. FryChristopher S. FryDepartment of Rehabilitation Sciences, College of Health Sciences, University of Kentucky, Lexington, KentuckyPublished Online:15 Mar 2014https://doi.org/10.1152/ajpcell.00022.2014This is the final version - click for previous versionMoreSectionsPDF (263 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations skeletal muscle atrophy is characterized by an increase in protein degradation that is accomplished through a variety of cellular mechanisms. Significant attention has been given to the ubiquitin-proteasome pathway and to expression of the E3 ubiquitin ligases muscle-specific RING finger-1 (MuRF1) and atrogin-1/muscle atrophy F box (MAFbx) during muscle atrophy. The increased expression of these ubiquitin ligases has been documented in multiple models of muscle atrophy, and an attenuation of atrophy has been shown after their knockdown (2). Induction of MuRF1 and atrogin-1 expression by the transcription factor forkhead box O3a (FOXO3a) is an integral step in enhanced protein degradation through the ubiquitin-proteasome pathway; however, regulation of MuRF1/atrogin-1 expression can also occur through the binding of microRNAs.MicroRNAs are small, noncoding RNAs that regulate protein expression through translation inhibition and/or degradation of specific transcripts. Recent work identified microRNA-23a (miR-23a) as capable of binding MuRF1 and atrogin-1 transcripts and inhibiting their translation (10). Overexpression of miR-23a also protected muscles from atrophy in vitro and in vivo (10). miR-23a expression is regulated in part through the transcription factor cytoplasmic nuclear factor of activated T cells 3 (NFATc3), which is activated by the phosphatase calcineurin (Cn) (6). This holds true in skeletal muscle as well, with expression or reporter activity of miR-23a responsive to Cn activity and NFATc3 expression (1, 10). Inhibition of Cn activity following an atrophy-inducing stimulus such as dexamethasone administration leads to decreased expression of miR-23a, thus relieving repression of MuRF1/atrogin-1 expression by miR-23a. Together with increased transcription through FOXO3a, this mechanism provides for increased expression of these E3 ubiquitin ligases, leading to enhanced degradation of proteins through the ubiquitin-proteasome pathway.In addition to regulation of microRNA at the transcriptional level, microRNA levels may also be controlled by their packaging into extracellular vesicles. Exosomes are small (30- to 100-nm) vesicles released from cells that contain proteins, mRNA, and microRNA. A recent study characterized exosomes released from C2C12 myoblasts, focusing primarily on protein content (3). Exosomes have also been suggested to serve as paracrine factors, whereby an exosome released from a donor cell can travel and fuse with a target cell, releasing its contents, which can influence activity of the target cell (9). This novel hypothesis is supported by a study demonstrating that microRNAs present in exosomes released by dendritic cells are capable of repressing mRNA translation in target cells (7). The packaging and subsequent release of microRNA-containing exosomes by donor cells could represent a new mechanism through which cells are capable of regulating microRNA levels.In this issue of American Journal of Physiology-Cell Physiology, Hudson et al. (4) report on two mechanisms through which skeletal muscle cells regulate intracellular levels of miR-23a: 1) Cn-NFATc3 transcriptional control and 2) rapid packaging and release into exosomes (4) (Fig. 1). The authors take a multifaceted approach in demonstrating transcriptional regulation of miR-23a through Cn. In a rat diabetic model of muscle atrophy, Cn/NFATc3 signaling was depressed, resulting in decreased levels of miR-23a (4). These findings provide evidence for an association of Cn activity and miR-23a levels on which the authors expand to demonstrate a direct relationship. Pharmacological activation of Cn in C2C12 myotubes increased miR-23a expression, and this increase in miR-23a expression was prevented with the addition of cyclosporine A to inhibit Cn. Knockout of either catalytic subunit of Cn in primary mouse myotubes led to decreased levels of miR-23a, and further reductions in miR-23a levels were prevented with the addition of dexamethasone, demonstrating Cn specificity in the expression of miR-23a (4). The authors make an interesting observation that constitutive activation of Cn does not increase miR-23a expression but does offer protection from dexamethasone-induced reduction of miR-23a expression (4). This finding is consistent with previous work (10) and provides evidence that a narrow range of Cn activity is required to regulate miR-23a levels. The breadth of experimental approaches employed by Hudson and colleagues demonstrates a direct link between Cn signaling and miR-23a expression.Fig. 1.miR-23a regulation via calcineurin/nuclear factor of activated T cells (NFAT) signaling and exosome incorporation during skeletal muscle atrophy. During atrophy-inducing conditions in muscle, a downregulation of calcineurin/NFAT signaling leads to a reduction of microRNA-23a (miR-23a) levels. Increased incorporation of miR-23a into exosomes further contributes to the decrease in miR-23a content during muscle atrophy. Increase in intracellular and exosomal miR-1 content, along with the contrasting intracellular and exosomal levels of miR-23a, demonstrates specificity of microRNA incorporation into exosomes. Downregulation of transcription and increased packaging into exosomes provide a 2-pronged approach for a reduction of miR-23a content during muscle atrophy.Download figureDownload PowerPointHudson et al. (4) propose a novel hypothesis that miR-23a levels are also regulated in part through the release of exosomes containing miR-23a. While exosomes have been shown to contain viable microRNAs capable of influencing protein expression in target cells (7), little is known regarding microRNA exosomal incorporation, and previous work in C2C12 myoblasts focused heavily on the protein content of exosomes (3). Hudson et al. demonstrate for the first time that dexamethasone treatment of C2C12 myotubes results in the release of exosomes enriched with microRNA (miR-1 and miR-23a). Exosomal microRNA content, not total number of exosomes, was increased with dexamethasone treatment, providing exciting evidence for the specificity of microRNA packaging into exosomes under differing physiological conditions. The increase in exosomal miR-23a content mirrors the decrease in intracellular content, which illustrates a new mechanism through which muscle cells can quickly reduce the content of specific microRNAs (4). Greater evidence for specific exosomal microRNA packaging is provided with miR-1. After dexamethasone treatment, intracellular and exosomal levels of miR-1 increase, whereas intracellular levels of miR-23a decrease, in accordance with an increase in exosomal levels of miR-23a (4) (Fig. 1). These data provide the first evidence for specificity of microRNA incorporation into exosomes under different physiological conditions. Future studies may target the secretion of exosomes to assess the contribution of exosome incorporation in the regulation of intracellular miR-23a levels. Exosome secretion requires production of ceramide by the enzyme neutral sphingomyelinase-2 (5, 8), and addition of neutral sphingomyelinase-2 inhibitors (manumycin-A and GW4869) will result in reduced secretion of exosomes, so the role of exosome incorporation of miR-23a can be tested under conditions similar to those presented by Hudson and et al.After an atrophy-inducing stimulus such as dexamethasone treatment, muscle cells may work to rapidly decrease miR-23a levels through decreased transcription via Cn/NFATc3 signaling and through the release of miR-23a in exosomes (4). The regulatory events governing the incorporation of microRNA into exosomes is largely unknown and warrants further investigation. The study by Hudson et al. (4) should help foster further interest in the role of exosomes and their transport of microRNA in skeletal muscle.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author.AUTHOR CONTRIBUTIONSC.S.F. prepared the figure, drafted the manuscript, edited and revised the manuscript, and approved the final version of the manuscript.REFERENCES1. Allen DL, Loh AS. Posttranscriptional mechanisms involving microRNA-27a and b contribute to fast-specific and glucocorticoid-mediated myostatin expression in skeletal muscle. Am J Physiol Cell Physiol 300: C124–C137, 2011.Link | ISI | Google Scholar2. Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA, Poueymirou WT, Panaro FJ, Na EQ, Dharmarajan K, Pan ZQ, Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD, Glass DJ. Identification of ubiquitin ligases required for skeletal muscle atrophy. Science 294: 1704–1708, 2001.Crossref | PubMed | ISI | Google Scholar3. Guescini M, Guidolin D, Vallorani L, Casadei L, Gioacchini AM, Tibollo P, Battistelli M, Falcieri E, Battistin L, Agnati LF, Stocchi V. C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction. Exp Cell Res 316: 1977–1984, 2010.Crossref | PubMed | ISI | Google Scholar4. Hudson MB, Woodworth-Hobbs ME, Zheng B, Rahnert JA, Blount MA, Gooch JL, Searles CD, Price SR. miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export. Am J Physiol Cell Physiol (December 11, 2013). doi:10.1152/ajpcell.00266.2013.Link | ISI | Google Scholar5. Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T. Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem 285: 17442–17452, 2010.Crossref | PubMed | ISI | Google Scholar6. Lin Z, Murtaza I, Wang K, Jiao J, Gao J, Li PF. miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy. 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Wada S, Kato Y, Okutsu M, Miyaki S, Suzuki K, Yan Z, Schiaffino S, Asahara H, Ushida T, Akimoto T. Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy. J Biol Chem 286: 38456–38465, 2011.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: C. S. Fry, Dept. of Rehabilitation Sciences, College of Health Sciences, Univ. of Kentucky, Lexington, KY 40503 (e-mail: christopher.[email protected]edu). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Cited BymiRNA-23a/27a attenuates muscle atrophy and renal fibrosis through muscle-kidney crosstalk26 March 2018 | Journal of Cachexia, Sarcopenia and Muscle, Vol. 9, No. 4Systems toxicology meta-analysis of in vitro assessment studies: biological impact of a candidate modified-risk tobacco product aerosol compared with cigarette smoke on human organotypic cultures of the aerodigestive tract1 January 2017 | Toxicology Research, Vol. 6, No. 5miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated exportMatthew B. Hudson, Myra E. Woodworth-Hobbs, Bin Zheng, Jill A. Rahnert, Mitsi A. Blount, Jennifer L. Gooch, Charles D. Searles, and S. Russ Price15 March 2014 | American Journal of Physiology-Cell Physiology, Vol. 306, No. 6 More from this issue > Volume 306Issue 6March 2014Pages C529-C530 Copyright & PermissionsCopyright © 2014 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.00022.2014PubMed24452375History Published online 15 March 2014 Published in print 15 March 2014 Metrics

Wnt/β-catenin signaling regulates follicular development by modulating the expression of Foxo3a signaling components

Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of β-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.

MiR-153 Supports Colorectal Cancer Progression via Pleiotropic Effects That Enhance Invasion and Chemotherapeutic Resistance

Although microRNAs (miRNA) have been broadly studied in cancer, comparatively less is understood about their role in progression. Here we report that miR-153 has a dual role during progression of colorectal cancer by enhancing cellular invasiveness and platinum-based chemotherapy resistance. miRNA profiling revealed that miR-153 was highly expressed in a cellular model of advanced stage colorectal cancer. Its upregulation was also noted in primary human colorectal cancer compared with normal colonic epithelium and in more advanced colorectal cancer stages compared with early stage disease. In colorectal cancer patients followed for 50 months, 21 of 30 patients with high levels of miR-153 had disease progression compared with others in this group with low levels of miR-153. Functional studies revealed that miR-153 upregulation increased colorectal cancer invasiveness and resistance to oxaliplatin and cisplatin both in vitro and in vivo. Mechanistic investigations indicated that miR-153 promoted invasiveness indirectly by inducing matrix metalloprotease enzyme 9 production, whereas drug resistance was mediated directly by inhibiting the Forkhead transcription factor Forkhead box O3a (FOXO3a). In support of the latter finding, we found that levels of miR-153 and FOXO3a were inversely correlated in matched human colorectal cancer specimens. Our findings establish key roles for miR-153 overexpression in colorectal cancer progression, rationalizing therapeutic strategies to target expression of this miRNA for colorectal cancer treatment.

IGF-1-Induced Enhancement of PRNP Expression Depends on the Negative Regulation of Transcription Factor FOXO3a

The conformational conversion of the cellular prion protein (PrPC) into its β-sheet-rich scrapie isoform (PrPSc) causes fatal prion diseases, which are also called transmissible spongiform encephalopathies (TSEs). Recent studies suggest that the expression of PrPC by the PRNP gene is crucial for the development of TSEs. Therefore, the identification of the exogenous and endogenous stimulating factors that regulate PRNP expression would help to understand the pathogenesis of TSEs. Here, we demonstrate that forkhead box O3a (FOXO3a) negatively regulates PRNP expression by binding to the PRNP promoter, which is negatively regulated by insulin-like growth factor 1 (IGF-1). Our results show that the IGF-1-induced enhancement of PRNP mRNA and protein levels is due to the activation of the PI3K-Akt signaling pathway. The activation of Akt then induces the phosphorylation of FOXO3a, leading to its translocation from the nucleus to the cytoplasm and preventing its binding to the PRNP promoter. Treatment with the PI3K-Akt inhibitor LY294002 induces the nuclear retention of FOXO3a, which leads to a decrease in PRNP expression. We present a new IGF-1-PI3K-Akt-FOXO3a pathway, which influences PRNP expression. The results of this work are vital for understanding the function of PrPC and for future therapeutic approaches to human TSEs.